MRE11, RAD50 and NBS1 form a highly conserved protein complex (the MRE11 complex) that is involved in the detection, signalling and repair of DNA damage. We identify MDC1 (KIAA0170/NFBD1), a protein that contains a forkhead-associated (FHA) domain and two BRCA1 carboxy-terminal (BRCT) domains, as a binding partner for the MRE11 complex. We show that, in response to ionizing radiation, MDC1 is hyperphosphorylated in an ATM-dependent manner, and rapidly relocalizes to nuclear foci that also contain the MRE11 complex, phosphorylated histone H2AX and 53BP1. Downregulation of MDC1 expression by small interfering RNA yields a radio-resistant DNA synthesis (RDS) phenotype and prevents ionizing radiation-induced focus formation by the MRE11 complex. However, downregulation of MDC1 does not abolish the ionizing radiation-induced phosphorylation of NBS1, CHK2 and SMC1, or the degradation of CDC25A. Furthermore, we show that overexpression of the MDC1 FHA domain interferes with focus formation by MDC1 itself and by the MRE11 complex, and induces an RDS phenotype. These findings reveal that MDC1-mediated focus formation by the MRE11 complex at sites of DNA damage is crucial for the efficient activation of the intra-S-phase checkpoint.

Mitotic fragmentation of the Golgi apparatus can be largely explained by disruption of the interaction between GM130 and the vesicle-docking protein p115. Here we identify a single serine (Ser-25) in GM130 as the key phosphorylated target and Cdc2 as the responsible kinase. MEK1, a component of the MAP kinase signaling pathway recently implicated in mitotic Golgi fragmentation, was not required for GM130 phosphorylation or mitotic fragmentation either in vitro or in vivo. We propose that Cdc2 is directly involved in mitotic Golgi fragmentation and that signaling via MEK1 is not required for this process.

Fragmentation of the mammalian Golgi apparatus during mitosis requires the phosphorylation of a specific subset of Golgi-associated proteins. We have used a biochemical approach to characterize these proteins and report here the identification of golgin-84 as a novel mitotic target. Using cryoelectron microscopy we could localize golgin-84 to the cis-Golgi network and found that it is enriched on tubules emanating from the lateral edges of, and often connecting, Golgi stacks. Golgin-84 binds to active rab1 but not cis-Golgi matrix proteins. Overexpression or depletion of golgin-84 results in fragmentation of the Golgi ribbon. Strikingly, the Golgi ribbon is converted into mini-stacks constituting only ∼25% of the volume of a normal Golgi apparatus upon golgin-84 depletion. These mini-stacks are able to carry out protein transport, though with reduced efficiency compared with a normal Golgi apparatus. Our results suggest that golgin-84 plays a key role in the assembly and maintenance of the Golgi ribbon in mammalian cells.

We have identified a protein motif, related to the zinc ringer, which dermes a newly discovered family of proteins. The motif was found in the sequence of the human RINGI gene, which is proximal to the major histocompatibility complex region on chromosome six. We propose naming this motif the "RING ringer" and it is found in 27 proteins, all of which have putative DNA binding functions. We have synthesized a peptide corresponding to the RING1 motif and examined a number ofproperties, including metal and DNA binding. We provide evidence to support the suggestion that the RING finger motif is the DNA binding domain of this newly defined family of proteins.Protein-DNA interactions are involved in many of the fundamental processes that occur inside cells, including transcription, replication, recombination, and restriction. To meet such a large number of functional requirements, a number of protein sequence and/or structural motifs have evolved that allow both specific and nonspecific DNA interaction (1, 2). Among these motifs are zinc fingers, which are autonomously folding domains that require zinc for folding and DNA binding activity (3, 4).The classic zinc finger motif is characterized by two conserved cysteines and histidines, which bind tetrahedrally to a zinc atom thereby stabilizing the secondary structure comprising an antiparallel two-stranded S-sheet and an a-helix (5, 6). The N-terminal end of the a-helix is responsible for making DNA sequence-specific interactions, with each finger recognizing 3 bp (7) as suggested previously (8). A second class of zinc finger has been described in the steroid/nuclear receptor family of proteins, which differs from the classic zinc finger in that the motif binds two zinc atoms to form a single folded domain with four cysteine ligands for each zinc (for review, see ref. 9). The mode of DNA interaction of this motif differs from that of the classic zinc finger in that the receptor binds as a dimer to a palindromic DNA sequence (10). However, sequence-specific DNA interactions are achieved by placing an a-helix in each of the major grooves ofthe DNA site (10). Recently, a third class of zinc finger fold has been described in the GAL4 DNA binding domain (11-13). GAL4 binds two zinc atoms through six cysteines, with the metals sharing two of the ligands (14). GAL4 also uses an a-helix in the major groove for sequence-specific DNA binding (11), which appears to be a common feature of the three zinc-mediated folds characterized to date.Previously, we have reported the identity of a cysteine-rich motif found in the sequence of the RING] gene that is related to the zinc finger (15). Here, we report the full sequence of the human RING] gene and define further this motif, which we propose to call the "RING finger." We extend the family of proteins containing the motif and characterize a synthetic peptide corresponding to the motif from RING1 in terms of zinc and DNA binding.tt MATERIALS AND METHODS DNA Sequencing. Sequencing reactions were performed by the primed synthesis...